Editing profiling of pde8a pre -mrna: use as specific biomarker of adars activities in human tissues to diagnose and to predict and assess therapeutic efficacy and/or efficiency or potential drug side effects

ABSTRACT

The present invention relates to the use of the editing profile of PDE8A pre-mRNA as a specific bio marker of ADARs activities in evolved primate, particularly in Human tissues. The present invention also relates to an in vitro method for predicting in Human an alteration of the mechanism of the ADARs catalysed pre-mRNA editing of target genes, by analysing the PDE8A pre-mRNA editing profile in a peripheral tissue sample containing cells expressing said PDE8A pre-mRNA, such as blood sample. The present invention is also directed to an in vitro method for the screening of potential therapeutic compound and to predict and assess therapeutic efficacy and/or efficiency or to diagnose potential severe brain or peripheral drug side effects implementing said PDE8A pre-mRNA editing profile as specific biomarker. The present invention is further directed to a method for determining the PDE8A pre-mRNA editing profile in Human, particularly by capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) method after amplification by a nested PCR. Finally the invention relates to particular nucleic acid primers implemented in said nested PCR and kit comprising such sets of primers and human cells capable of expressing PDE8A and ADARs.

The present invention relates to the use of the editing profile of PDE8Apre-mRNA as a specific biomarker of ADARs activities in evolved primate,particularly in Human tissues. The present invention also relates to anin vitro method for predicting in Human an alteration of the mechanismof the ADARs catalysed pre-mRNA editing of target genes, by analysingthe PDE8A pre-mRNA editing profile in a peripheral tissue samplecontaining cells expressing said PDE8A pre-mRNA, such as blood sample.The present invention is also directed to an in vitro method for thescreening of potential therapeutic compounds and to predict and assesstherapeutic efficacy and/or efficiency or to diagnose potential severebrain or peripheral drug side effects implementing said PDE8A pre-mRNAediting profile as specific biomarker. The present invention is furtherdirected to a method for determining the PDE8A pre-mRNA editing profilein Human, particularly by capillary electrophoresis single-strandconformation polymorphism (CE-SSCP) method after amplification by anested PCR. Finally the invention relates to particular nucleic acidprimers implemented in said nested PCR and kit comprising such sets ofprimers and human cells capable of expressing PDE8A and ADARs.Alternatively, PDE8A pre-mRNA editing profiling in Human could becarried out by ultra high-throughput sequencing (HTS) technologies.Abbas and collaborators in one hand, and Morabito and collaborators inthe other have recently developed editing quantification applicationsfor the HTS Illumina technology (Abbas et al., 2010, Nucl. Acid Res.;Morabito et al., 2010, Mol. Pharmacol.) (see material and methodssections of the cited articles). These approaches have been set up for5-HT2cR mRNA editing quantification in the mouse brain. The resultspresented by the authors are very close to those described by Poyau andcollaborators in their princeps paper (Poyau, 2007). With somemodifications of the design of primers used for cDNA amplification(second nested PCR), and of the size of the generated amplicons, theIllumina technology could also be applied to PDE8A pre-mRNA editinganalyzis. Similarly the HTS Ion Torrent semiconductor sequencingtechnology (Life technologies) based on direct electrical detection ofDNA synthesis could be used for PDE8A pre,-mRNA editing profilequantification (Pourmand et al., 2006. Proc. Natl. Acad. Sci.; Andersonet al., 2008, Sens. Actuators B. Chem.). Whatever the method used, thestarting material would remain the cDNA libraries built from total RNAsand the first PCR products described in the material and methods of thispatent.

Adenosine (A) to Inosine (I) RNA editing is accepted as a fundamentalpost transcriptional mechanism of control of a large population offunctional proteins. Due to the activities of specific enzymes (ADARs)it can control a large set of targets (1). It implicates the formationof pre-mRNAs double stranded structures on which specific edited siteshave been identified. These Adenosines can belong to a coding sequence(eg: 5-HT2c receptor (5HT2CR), Glutamate receptor) (2, 3) or an intronicsequence (Phosphodiesterase subtype 8A (PDE8A)(4, 5). In this laterconfiguration the editing process could influence both the RNA splicingpattern and/or the formation of different isoforms of miRNAs (6). Theobserved modifications of sequences due to the editing can thus be dueto modifications of the relative concentrations of the different ADARsat the editing processing site, and/or to an alteration of theefficiency of the editing enzymes themselves. Previous studies haveclearly indicated that both types of mechanisms could be at the originof specific pathological or pharmacological alterations (7, 5).

The invention presents the rapid and quantitative onset of the editingprofile of PDE8A pre-mRNA as a strongly efficient tools to predictsignificant alterations of the activities of ADARs in total RNA extractsof Human tissues including Brain and of more easily accessible bloodsamples. Genetic observations indicate that the rapid solution of thisgenomic approach opens new possibilities to evaluate primary orsecondary risks of severe psychiatric drug induced side effects.

The PDE8A gene is located on chromosome 15q25.3 (Ensemble Genomebrowser). A genome scan using 297 families with proband who hadrecurrent early-onset major depressive disorder (MDD) observedsignificant evidence for linkage on chromosome 15q25 .3-q26.2 (8). Theauthors confirmed this genetic association in their final report (9).The most significant scores were observed for the markers D15S652 andGATA128A02. An independent genome scan of 497 sib pairs with recurrentdepression found a modest signal for linkage at the same position onchromosome 15q, with the most significant marker (D15S1047) locatedapproximately 11 Mb proximal to D15S652 (10). Another genome scan, usingpedigrees with recurrent early-onset depression and anxiety disorders,found suggestive evidence for linkage to 15q in this same region (11).Further association studies trying to ascribe to the NTRK3 gene (locatedapproximately 5 Mb proximal to D15S652) a role in MDD were not fullyconvincing (11, 12). The authors suggest that this gene is probably nota major contributor to the overall risk for depression.

Recently, a meta-analysis of 3 European-ancestry MDD genome-wideassociation study data sets was carried out by Shyn and collaborators(Shyn et al., 2011, Mol. Psychiatry). The data sets totalized 3957 casesand 3428 controls. Among several candidates, the authors found that SNPrs11634319 (chr15: 84,224,912) located in band 15q25.3 in the vicinityof the PDE8A gene was associated to a female-narrow phenotype of majordepression (p<10⁻⁵).

On the other hand, older experiments conducted by Aston andcollaborators, had compared gene expression in the temporal cortex from12 patients with major depressive disorder and 14 matched controls byusing Affimetrix microarrays (Aston et al., 2005, Mol. Psychiatry).Significant expression changes were identified in families of genesinvolved in neurodevelopment, cell communication and signaltransduction. Among the latter family, the mRNA abundance of the PDE8Agene was twofold less in depressed people (p<0.0003). This result washighly significant as the PDE8A mRNAs were detected in all controls andpatients unlike messengers of other genes. All these linkage,association, and microarray gene expression studies tend to indicatethat the PDE8A gene could be associated to major depression disorder.

Using an original method based on glyoxilated poly(A)+ RNA treated byRNase T1, Morse and collaborators have identified new targets of theediting enzymes ADARs in the Human brain (4). They showed thatnon-coding regions of pre-mRNA (introns, 3′UTR) appear to be the primarytargets of ADARs. Intron 9 of the PDE8A gene (13) (PDE8A gene imbeddedin contig NT_010274) coding for a c-AMP specific phosphodiesterase isone of these targets. Recently, in the scope of systemic autoimmunelupus erythematosus disorder, and in T-lymphocytes, Orlowski andcollaborators have observed two hot spots for A to I editing in PDE8Agene transcripts (5). The first one, called site 1, concerned twoadenosine residues at positions 5505 and 5506 of intron 9. We calledthese editing sites A and B, respectively. The second hot spot or site 2concerned three other adenosines at positions 5536, 5538 and 5539 ofintron 9. These editing sites were called C, D and E, respectively. Lowfrequency sites of editing were also observed at base positions 5468,5548 and 5617 of intron 9 of gene PDE8A (editing sites H, F and G,respectively). All these editing sites, embedded in their sequencecontexts are shown in FIG. 1.

The 2D structure of RNA sequence corresponding to base positions 5367 to5736 of intron 9 of gene PDE8A is presented in FIGS. 2 and 3. Onlyediting sites A, B, F, G and H are embedded in stem structures, C, D andE are in a loop.

There is a need to provide with in vitro tests which can rapidlydetermine the activity of the ADARs, particularly a specific biomarkerof these ADARs activities in Human tissues easy to analyse. Such testswill allow the demonstration of alteration of the ADARs catalysed-pre-mRNA editing mechanism.

A platform implementing such a specific ADARs biomarker activities couldbe proposed to evaluated, for example at a pre-clinical stage, thepotential effect of new therapeutic molecules on the editing regulationassociated to this ADARs catalyzed -pre-mRNA editing mechanism sinceediting has been already found altered in patients suffering fromdepression or having committed suicide. Moreover, such a specific ADARsbiomarker activities could be also proposed for rapid, effective methodsby which we can diagnose in patients pathologies associated to thealteration of this editing mechanism or by which we can determine thepotential toxicity of efficiency of test compounds, or potentialside-effects profile of a drug in man, particularly before thepost-marketing period. There is also a need for tools and kits for theimplementation of such methods.

This is the object of the present invention.

The inventors have demonstrated that editing alterations produced byincreasing concentrations of IFNα applied to SH-SY5Y human cells couldinfluence the steady state of PDE8A protein isoforms. They have thusobserved after a western blot analysis of the PDE8A proteinsconcentrations in the SH-SY5Y cells, that the concentration of PDE8A4isoform of the protein could be significantly (R²=0.82) and negativelylinearly correlated (slope=−3.6) with the positive Δ of variation of theRQ versus GAPDH of the ADAR1a-p150 induced by IFNα application during 24hours. The best fitting of the effect versus concentrations of IFNα ledto an estimated EC 50% of 3.3±4.4 (SD) IU/ml with a R² of the fit=0.7and a corresponding maximal effect of −47% of the steady stateconcentration found in controls.

This example demonstrates that the editing process would preciselycontrol the expression of the PDE8A proteins either by its possibleinfluence on the splicing mechanism or by the control of the sequence ofdedicated miRNAs.

Moreover, the major interest of this specific editing target is that itis present particularly in the Human Blood as in brain which is not thecase of 5-HT2CR. The editing profile of PDE8A can be thus considered asa specific tool to evaluate the activity of the editing enzymesmachinery in human tissues including blood.

Together to the analysis of the complete editing profile of the PDE8A inevolved primate including Human, the inventors have demonstrated that anevaluation of the expression of the editing profile of the PDE8Apre-mRNA as a complementary approach is particularly well adapted to theevaluation of the general editing context of dysregulation of theediting machinery. It includes the quantitative and qualitative analysisof the editing profile of the PDE8A pre-mRNA (particularly byquantitative nested RT-PCR associated to a CE-SSCP method).

The examples below strongly support the interest of measuring theediting process of this specific target as an innovative tool in variouspreclinical and clinical investigations.

Thus, the present invention is directed to a method in vitro for thedetermination of the ADARs (Adenosine Deaminase, RNA-specific) activitycomprising the following steps of:

-   a) obtaining a biological sample containing mammal cells wherein    said mammal cells express the editing enzymes ADAR1a, ADAR1b and    ADAR2 and the phosphodiesterase subtype 8A (PDE8A);-   b) determining in a cellular extract the editing profile of the    PDE8A pre-mRNA measured in a cellular RNA extract obtained from said    cellular extract, preferably said editing profile giving the mean    proportion of each identified isoform of the PDE8A pre-mRNA;-   c) comparing the profile obtained in step b) between said mammals    cells with the PDE8A pre-mRNA editing profile obtained for control    cells whose ADARs activity is known.

In another aspect, the present invention is directed to an in vitromethod for identifying in vitro or to diagnose whether a patientpresents a pathology or is at risk to develop a pathology related to analteration of the ADARs catalyzed pre-mRNA editing mechanism, whereinthis method comprising the following steps of:

-   a) obtaining from the patient to be tested a biological sample    containing cells wherein said cells express the editing enzymes    ADAR1a, ADAR1b and ADAR2 and the PDE8A, preferably a biological    sample containing PBMC (Peripheral Blood Mononuclear Cells);-   b) determining the editing profile of the PDE8A pre-mRNA measured in    a cellular RNA extract obtained from said biological sample    containing cells, preferably said editing profile giving the mean    proportion of each identified isoform of the PDE8A pre-mRNA;-   c) identifying whether said patient presents or is at risk to    develop such a pathology by comparing the editing profile of the    PDE8A pre-mRNA obtained in step b) with control editing profile of    the PDE8A pre-mRNA obtained for normal patients and/or for patients    exhibiting pathologies related to an alteration of the mechanism of    this mRNA editing.

In a preferred embodiment, a significant difference between the editingprofiles of the PDE8A pre-mRNA measured between the control sample andthe tested patient sample is indicative of the presence of such apathology or a risk for such a pathology.

By significant difference, it is intended to designate a difference ofat least 2%, preferably at least 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10%between the tested patient cells and the control cells for at least oneof the same isoform (edited or non edited isoform), more preferably thisat least one of the same isoform being selected from the group of theAB, ABC, ABE, ABEF, ABEFG, ABG, B, BC, BD, BE, BEG, BF, BFG, BG, M orthe non edited isoform (ned or ne or Ne), the most preferred being theB, AB, BC, ABC and ned isoform.

In a more preferred embodiment, by significant difference, it isintended to designate a difference of at least 2%, preferably at least3%, 4%, 5%, 6%, 7%, 8%, 9% and 10% between the tested patient cells andthe control cells for at least a same pair of isoforms (edited or nonedited isoform), more preferably this at least same pair of isoformsbeing selected from the group of the AB, ABC, ABE, ABEF, ABEFG, ABG, B,BC, BD, BE, BEG, BF, BFG, BG, M edited isoforms and the non editedisoform (ned), the most preferred being a same pair selected in thegroup consisting of the B, AB, BC, ABC and ned isoforms, B and nedisoforms being the most preferred pair of isoforms.

In another aspect, the invention is directed to an in vitro method formonitoring the efficacy of a therapeutic treatment in a patient having apathology or at risk to present a pathology related to an alteration ofthe ADARs catalyzed pre-mRNA (or mRNA) editing mechanism, said methodcomprising the step of:

-   a) obtaining a biological sample from said patient before the    treatment and at least after one interval during said treatment and    wherein said biological sample contains cells, said cells expressing    the editing enzymes ADAR1a, ADAR1b and ADAR2 and PDE8A;-   b) determining in a cellular extract from said biological sample the    editing profile of the PDE8A pre-mRNA measured in a cellular RNA    extract obtained from said cellular extract, preferably said editing    profile giving the mean proportion of each identified isoform of the    PDE8A pre-mRNA;-   c) comparing the results obtained in step b) between said biological    sample cellular extract obtained from the patient before and after    said interval during the treatment.

In a preferred embodiment, a non-significant modification of said PDE8Apre-mRNA editing profile measured after said interval during thetreatment is indicative of a lack efficacy of the therapeutic treatment.

In an also preferred embodiment, the obtaining of a PDE8A pre-mRNAediting profile measured after said interval having significantly thesame profile obtained for biological sample controls from subjects whodo not present or who are not at risk to present a pathology related toan alteration of the ADARs catalyzed pre-mRNA (or mRNA) editingmechanism, is indicative of an efficacy of the treatment.

In another aspect, the present invention comprises a method in vitro forthe determination of the potential toxicity or side-effects of a testcompound after its administration in a patient and wherein said compoundis tested for its ability to prevent or to treat a pathology directly orindirectly related to an alteration of the ADARs catalyzed pre-mRNA (ormRNA) editing mechanism comprising the following steps of:

-   a) obtaining a biological sample containing mammal cells wherein    said mammal cells express the editing enzymes ADAR1a, ADAR1b and    ADAR2 and the phosphodiesterase subtype 8A (PDE8A);-   b) contacting said mammals cells with the compound to be tested;-   c) determining in a cellular extract the editing profile of the    PDE8A pre-mRNA measured in a cellular RNA extract obtained from said    cellular extract, preferably said editing profile giving the mean    proportion of each identified isoform of the PDE8A pre-mRNA;-   d) comparing the results obtained in step c) between said treated    cells with the compound to be tested obtained in step b) and non    treated control cells.

In the context of the invention, the term “toxicity” refers to anyadverse and/or side effect of a compound on the metabolism of a cell ora tissue and more generally any alteration in metabolism that can resultin a harmful effect of the compound on the patient, particularly in thecontext of the present invention the potential risk of drug induced mooddisturbance and suicide.

The term “test compound” refers in general to a compound to which a testsubject is exposed. Typical test compounds will be small organicmolecules, typically drugs and/or prospective pharmaceutical leadcompounds, but can include proteins, peptides, polynucleotides,heterologous genes (in expression systems), plasmids, polynucleotideanalogs, peptide analogs, lipids, carbohydrates, viruses, phage,parasites, and the like.

The term “control compound” refers to a compound that is not known toshare any biological activity with a test compound, which is used in thepractice of the invention to contrast “active” (test) and “inactive”(control) compounds during the derivation of Group Signatures and DrugSignatures. Typical control compounds include, without limitation, drugsused to treat disorders distinct from the test compound indications,vehicles, inactivated versions of the test agent, known inert compounds,and the like.

In another aspect, the present invention encompasses a method in vitrofor the selection of a therapeutical compounds useful for the treatmentof pathology related to an alteration of the ADARs catalyzed pre-mRNA(or mRNA) editing mechanism (ADAR dependent A to I pre-mRNA editingmechanism) comprising the following steps of:

-   a) obtaining a biological sample containing mammal cells wherein    said mammal cells express the editing enzymes ADAR1a, ADAR1b and    ADAR2 and PDE8A:-   b) contacting said mammals cells with the compound to be tested:-   c) determining in a cellular extract the editing profile of the    PDE8A pre-mRNA measured in a cellular RNA extract obtained from said    cellular extract, preferably said editing profile giving the mean    proportion of each identified isoform of the PDE8A pre-mRNA;-   d) comparing the results obtained in step c) between said treated    cells with the compound to be tested and non treated control cells.

In a preferred embodiment of the methods of the present invention instep b) said mammals cells are cultured in presence of the compound tobe tested in a medium suitable or convenient for the culture of saidmammal cells.

Preferably, in step b) said mammals cells are cultured in presence ofthe compound to be tested for at least the time necessary to modify theexpression of edited isoforms of PDE8A pre-mRNA and/or the ADAR1a,ADAR1b and ADAR2 enzymes expressed, whether they can be modified by sucha compound.

Preferably in step b) said mammals cells are cultivated in presence ofthe compound to be tested for at least 1 hour, more preferably at least5, 10, 24 or 48 hours before the step c) of determining in the samecellular extract the editing profile of each identified isoform of thePDE8A pre-mRNA and/or the quantitative expression of said editingenzymes ADAR1a, ADAR1b and ADAR2.

In a preferred embodiment of the methods of the present invention thecompound to be tested is further administered in vivo to an evolvedprimate animal model, suitable to test the same compound and wherein thepotential toxicity or side-effects of this test compound after itsadministration in this animal model can be evaluated, particularly byevaluating the alteration of the pre-mRNA editing of the PDE8A and/orthe ADAR isoforms expressed in total blood, particularly PBMC, or inbrain.

In a preferred embodiment, in addition to the determination of theediting profile of the PDE8A pre-mRNA measured in a cellular RNA extractin step b) or c) of the methods according to the invention, these stepsfurther comprises the determination of the quantitative expression ofsaid editing enzymes ADAR1a, ADAR1b and ADAR2 in a cellular extract ofsaid cells.

The step of comparing the results obtained in step c) or d) furthercomprises the comparison of the results obtained between the treatedcells and non treated control cells for ADAR expression.

In a preferred embodiment, the editing profile of the PDE8A pre-mRNAmeasured in the cellular RNA extract and the quantitative expression ofsaid editing enzymes ADAR1a, ADAR1b and ADAR2 are determined in the samecellular extract.

In an also preferred embodiment, the quantitative expression of saidediting enzymes ADAR1a, ADAR1b and ADAR2 is determined by the measure ofthe mRNA expression of said editing enzymes or by the measure of saidediting enzymes protein expressed in the cellular extract.

In another preferred embodiment, the quantitative expression of saidediting enzymes ADAR1a, ADAR1b and ADAR2 is determined by the measure ofthe mRNA expression of said editing enzymes, preferably determined inthe same total RNA cell extract used for the determination of theediting profile of the PDE8A pre-mRNA.

In another preferred embodiment, said mammal cells capable of expressingthe ADARs isotypes ADAR1a, ADAR1b and ADAR2, and the PDE8A are from anevolved primate, such as chimpanzee, Rhesus macaque or Human.

In another preferred embodiment, said mammal cells are human cells orhuman derived cells such as cells derived from human cell line orrecombinant cells originating from human cell line.

In method according to one of claims 1 to 12, wherein in step a), saidmammal cells capable of expressing the ADARs isotypes ADAR1a, ADAR1b andADAR2, and the PDE8A are from a biological sample of a subject or apatient selected from the group of biological samples consisting inblood samples (PBMC) or body fluids (LCR) or preoperative and postmortem tissues (brain, liver, prostate) or biopsy samples (skin,tumors).

In another preferred embodiment, said mammal cells capable of expressingthe ADARs isotypes ADAR1a, ADAR1b and ADAR2, and the PDE8A are from abiological sample containing cells derived from a cell line.

In another preferred embodiment, said mammal cells capable of expressingthe ADARS isotypes ADAR1a, ADAR1b and ADAR2, and the PDE8A are from abiological sample containing cells derived from a cell line selectedfrom the group consisting of evolved primate neuroblastoma,glioblastoma, astrocytoma and other tissue or tumor specific cell lines,preferably from the human neuroblastoma SH-SY5Y cell line.

In another preferred embodiment, said mammal cells capable of expressingthe ADARs isotypes ADAR1a, ADAR1b and ADAR2, and the PDE8A are from abiological sample containing cells derived from a recombinant cell line,optionally transformed by a vector carrying a nucleic acid encoding forat least one of an evolved primate ADARs isotypes selected from ADAR1a,ADAR1b and ADAR2 isotypes, or an evolved primate PDE8A.

In a preferred embodiment, said pathology related to an alteration ofthe ADARs catalyzed pre-mRNA editing mechanism, is a pathology selectedfrom the pathologies related to an alteration of:

-   the ADARs catalyzed 5-HT2c receptor (5-HT2CR) mRNA editing    mechanism;-   the ADARs catalyzed GluR-B receptor pre-mRNA editing mechanism;-   the ADARs catalyzed K(V)1⋅1 potassium channel mRNA editing    mechanism,-   the ADARs catalyzed FlnA or Blcap (Riedmann et al; RNA 2008, 14:    1110-111 8 Li et al; Science 2009 324, 1210)) mRNA editing    mechanism;-   ADARs catalyzed viral RNAs after infection mRNA editing mechanism.

In a more preferred embodiment, wherein said pathology related to analteration of the ADARs catalyzed pre-mRNA (or mRNA) editing mechanismis selected from the group consisting of psychiatric, neurological,immunological and degenerative syndromes associated to an alteration ofPDE8A intronic A to I pre-mRNA editing

When said pathology is related to the ADARs catalyzed 5-HT2C receptormRNA editing mechanism is selected from the group consisting of mentaldisorders, schizophrenia, depression, Bipolar disease, suicide orabnormal feeding behaviour (obesity, anorexia), but also Mild CognitiveImpairement (MCI), Epilepsia, Alzheimer or Chronical pain syndromes.

When said pathology is related to the ADARs catalyzed GluR-B receptormRNA editing mechanism is selected from the group consisting in MCI,Epilepsia, Alzheimer, Amyotrophic Lateral Sclerosis (ALS) (Hideyama etal, 2010, J Pharmacol. Sci. 113, 9-13).

Additionally, related to ADAR1 genetic alterations the Dyschromatosissymetrica hereditaria (Suzuki et al., 2007, J invest. Dematol. 309-311.

In another aspect, said mammals cells implemented in the methods of thepresent invention further express the 5-HT2CR and, in addition to thedetermination of the editing profile of the PDE8A pre-mRNA measured in acellular RNA extract in step b) or c) of the methods according to theinvention, and, optionally comprising the determination of thequantitative expression of the editing enzymes ADAR1a, ADAR1b and ADAR2in a cellular extract of said cells, this step further comprises thedetermination of the editing profile of the 5-HT2CR mRNA, preferably bythe CE-SSCP method already described for the determination of theediting profile of the 5-HT2CR mRNA in human samples in the patentdocument PCT/EP2008/057519 filed on Jun. 13, 2008 and the patentdocument PCT/EP2009/067464 filed on Dec. 17, 2009.

When the editing profile of the 5-HT2CR mRNA is also determined, thestep of comparing the results obtained in step c) or d) furthercomprises the comparison of the results obtained between the treatedcells and non treated control cells for the editing profile of the5-HT2CR mRNA.

In a preferred embodiment, the editing profile of the PDE8A pre-mRNAmeasured in a cellular RNA extract obtained from said cellular extract,preferably said editing profile giving the mean proportion of eachidentified isoform of the PDE8A pre-mRNA comprises at least thedetermination and/or the quantification of the edited sites: A, B, C, D,E, F, G, H, I, J, K, L, M, N, or at least one of the PDE8A pre-mRNAisoforms comprising at least one of the editing sites A, B, C, D, E, F,G, H, I, J, K, L, M, N additionally with the non edited isoform (ned).

In a more preferred embodiment, the editing profile of the PDE8Apre-mRNA measured in a cellular RNA extract obtained from said cellularextract, preferably said editing profile giving the mean proportion ofeach identified isoform of the PDE8A pre-mRNA comprises at least thedetermination and/or the quantification of the PDE8A pre-mRNA isoformsAB, ABC, ABE, ABEF, ABEFG, ABG, B, BC, BD, BE, BEG, BF, BEG, BG, M orthe non edited isoform (ned).

In an also more preferred embodiment, the editing profile of the PDE8Apre-mRNA measured comprises at least the determination and/or thequantification of the isoforme B or at least one of the PDE8A pre-mRNAisoforms comprising at least the editing site B edited.

In an also more preferred embodiment, the editing profile of the PDE8Apre-mRNA measured comprises at least the determination and/or thequantification of the isoforms (ned).

In an also more preferred embodiment, the editing profile of the PDE8Apre-mRNA measured comprises at least the determination and/or thequantification of the isoforms ned and B, preferably at least (ned), Ban AB, more preferably at least (ned), B, AB and BC.

In a preferred embodiment of the method according to the presentinvention, the editing rate for each edited and unedited form of saidPDE8A pre-mRNA is determined by a method which comprises the followingsteps:

A) extraction of the total RNAs of said mammal cells, followed, whereappropriate, by purification of the pre-mRNAs;

B) reverse transcription of the RNAs extracted in step A); and

C) PCR amplification of the cDNAs obtained in step B) using at least apair of primers specific for the PDE8A pre-mRNA fragment containing theedition sites which may be edited, this pair of primers being chosen soas to be able to amplify all the editing forms and the unedited formpotentially present in the RNA extract.

In a preferred embodiment of the method according to the presentinvention, the editing rate for each edited and unedited form of saidPDE8A pre-mRNA is determined by a method which comprises the followingsteps:

A) extraction of the total RNAs of said mammal cells, followed, whereappropriate, by purification of the pre-mRNAs;

B) reverse transcription of the RNAs extracted in step A); and

C) PCR amplification of the cDNAs obtained in step B) using at least apair of primers specific for the PDE8A pre-mRNA fragment containing theedition sites which may be edited, this pair of primers being chosen soas to be able to amplify all the editing forms and the unedited formpotentially present in the RNA extract, and wherein the step B) ofreverse transcription is carried out by using an oligonucleotidic primerspecific of the PDE8A gene.

In a preferred embodiment of the method according to the presentinvention, in step C), the primers used in the PCR amplification step(in the second round if it is a nested type PCR) are labelled,preferably labelled with fluorophores.

In a preferred embodiment of the methods of the present invention instep c), the editing profile giving the mean proportion of eachidentified isoform of the PDE8A pre-mRNA is determined by an SSCP methodcapable of providing the editing profile for each of the edited andunedited separate forms of said pre-mRNA, said SSCP method beingcharacterized in that it comprises after the steps A), B) and C) thefollowing steps:

D) where appropriate, purification of the PCR products obtained in stepC);

E) where appropriate, quantification of the PCR products obtained instep D);

F) dissociation of the double-stranded cDNAs to single-stranded cDNAs,in particular by heating followed by abrupt cooling;

G) separation of the single-stranded cDNAs by capillary electrophoresis;and

H) obtaining of the editing profile by reading of the fluorescence and,where appropriate, acquisition of the profile data by means of theexploitation system associated with the fluorescence reader.

In a particular preferred embodiment, the editing profile, preferablythe editing profile giving the mean proportion of each identifiedisoform, of the PDE8A pre-mRNA measured in the cellular RNA extract ofevolved primate cells, preferably human, is measured by RT followed by anested type PCR comprising two rounds of PCR, and wherein:

-   a) the first round of PCR is carried out by the following sets of    primers:

Forward: PDE8A-1FWD (SEQ ID NO. 13) GCTGAAGCCTTCCTTCTAAGG Reverse:PDE8A-1REV (SEQ ID NO. 12) GGACCTAGAGTTGACCCAGGand wherein

-   b) the second round of PCR is carried out by the following set of    primers:

Forward: PDE8A-2FowFAM (SEQ ID NO. 10) CTAGGGAACCCTGTTTAGTCC Reverse:PDE8A-2Rev VIC (SEQ ID NO. 11) CAATGGGCACCAAAAAAGGG

When ADARs specific isoforms have to be determined in the method, it ispreferred that the the pair of primers specific for the evolved primate,preferably human, ADAR mRNA PCR amplification are selected from thegroup consisting of:

-   for ADAR1-150 isoform mRNA amplification:

Forward: (SEQ ID NO. 14) 5′-GCCTCGCGGGCGCAATGAATCC-3′ Reverse: (SEQ IDNO. 15) 5′-CTTGCCCTTCTTTGCCAGGGAG-3′

-   for ADAR1-110 isoform mRNA amplification:

Forward: (SEQ ID NO. 16) 5′-CGAGCCATCATGGAGATGCCCTCC-3′ Reverse: (SEQ IDNO. 17) 5′-CATAGCTGCATCCTGCTTGGCCAC-3′

-   for ADAR2 mRNA amplification:

Forward: (SEQ ID NO. 18) 5′-GCTGCGCAGTCTGCCCTGGCCGC-3′ Reverse: (SEQ IDNO. 19) 5′-GTCATGACGACTCCAGCCAGCAC-3′

In a more preferred embodiment of the method according to the presentinvention, the quantification of the editing profiles of PDE8A pre-mRNAincluding separation and identification of each of the isoforms iscarried out by capillary electrophoresis single strand conformationpolymorphism (CE-SSCP).

In a particular preferred embodiment, the method of the presentinvention further comprises a step of determining the quantity of thePDE8A mRNA expressed in said mammal cell by a Q-PCR or a step ofdetermining the quantity of the PDE8A polypeptide expressed in saidmammal cell.

In a preferred embodiment of the methods of the present invention instep b) or c), when the editing profile of the 5-HT2CR mRNA, preferablygiving the mean proportion of each identified isoform of the 5-HT2CRmRNA measured in the cellular RNA extract, is determined, saiddetermination is carried out by a nested type PCR comprising two roundsof PCR, and

-   wherein the first round of PCR is carried out by the following sets    of primers:

Forward: (SEQ ID NO. 20) 5′-TGTCCCTAGCCATTGCTGATATGC-3′, Reverse: (SEQID NO. 21) 5′-GCAATCTTCATGATGGCCTTAGTC-3′;and wherein the second round of PCR is carried out by the following setof primers:

Forward: (SEQ ID NO. 22) 5′-ATGTGCTATTTTCAACAGCGTCCATC-3′, Reverse: (SEQID NO. 23) 5′-GCAATCTTCATGATGGCCTTA-3′.

In another aspect, the present invention is directed to a kit for thedetermination of the editing profile of the PDE8A pre-mRNA comprising:

-   a) the following set of primers:

(SEQ ID NO. 13) GCTGAAGCCTTCCTTCTAAGG (SEQ ID NO. 12)GGACCTAGAGTTGACCCAGGand

-   b) the following set of primers, preferably labelled:

(SEQ ID NO. 10) CTAGGGAACCCTGTTTAGTCC (SEQ ID NO. 11)CAATGGGCACCAAAAAAGGG

The present invention also relates to a kit for the determination of theediting profile of the PDE8A pre-mRNA comprising:

-   a) mammal cells from evolved primate cell line, preferably human    cell line, wherein said cells express the editing enzymes ADAR1a,    ADAR1b and ADAR2 and the PDEA8, optionally the serotonin 2C receptor    (5HTR2C); and-   b) two set of primers for determining by a RT/PCR involving a nested    type PCR comprising two rounds of PCR the editing profile of the    PDE8A pre-mRNA which can be present in a RNA extract of said mammal    cells; and/or-   c) optionally a set of primers for measuring by a quantitative Q-PCR    the quantitative expression of the editing enzymes ADAR1a, ADAR1b    and ADAR2 and the level of expression of the PDE8A mRNA by using the    following set of primers (Applied Biosystems references):-   PDE8A1 Hs 01079628_m1-   PDE8A Hs 00400174_m1

In a preferred embodiment, said mammal cells are from an evolved primatetumor cell line, such as neuroblastoma, glioblastoma or astrocytoma cellline, preferably from a human neuroblastoma cell line, more preferablythe human neuroblastoma SH-SY5Y cell line.

In a more preferred embodiment the two sets of primers of b) in the kitof the invention are:

-   a) the following set of primers:

(SEQ ID NO. 13) GCTGAAGCCTTCCTTCTAAGG (SEQ ID NO. 12)GGACCTAGAGTTGACCCAGGand

-   b) the following set of primers, preferably labelled:

(SEQ ID NO. 10) CTAGGGAACCCTGTTTAGTCC (SEQ ID NO. 11)CAATGGGCACCAAAAAAGGG

Finally, in a particular aspect, the present invention is directed to anin vitro method for the determination or for the prediction of thepotential toxicity or side-effects of a interferon alpha (IFNα)treatment after its administration in a patient, particularly for apatient infected by the HCV (Hepatitis C virus), said method comprisingthe following steps of:

-   a) obtaining a biological sample containing mammal white cells,    preferably PBMC cells, from said treated patient;-   b) determining in the cellular extract of said biological sample    containing mammal white cells the quantitative expression of the    editing profile of the PDEA8 pre-mRNA, and optionally the editing    enzymes ADAR1a, ADAR1b and ADAR2, measured in the cellular RNA    extract;-   d) comparing the results obtained in step b) between said cells from    said IFNα treated patient with non treated control cells or with,    IFNα treated cells prior obtained from the same patient at the    beginning or during the IFNα treatment.

The following examples and also the figures and the legends hereinafterhave been chosen to provide those skilled in the art with a completedescription in order to be able to implement and use the presentinvention. These examples are not intended to limit the scope of whatthe inventor considers to be its invention, nor are they intended toshow that only the experiments hereinafter were carried out.

LEGEND TO THE FIGURES

FIG. 1: Partial sequence of intron 9 of the PDE8A gene and coordinatesof the edited adenosine residues. An internal sequence (432 bp) ofintron 9 (base positions 5305 to 5736) is presented. Previouslydescribed editing sites by Orlowski and collaborators (5) are in boldand their name depicted in black capital letters above the sequence(coordinates in intron 9 of the PDE8A gene: H=5468; A=5505; B=5506;C=5536; D=5538; E=5539; F=5548; G=5617).

FIG. 2: Overall putative stem and loop structure of RNA sequence ofintron 9 of the Human gene PDE8A (bases 5367 to 5736). The 2D RNAstructure is calculated by the KineFold program(http://kinefold.curie.fr/). Editing sites described by Orlowski andcollaborators are depicted in red (5).

FIGS. 3A and 3B: Zoom on editing sites A, B, C, D, F, F (3A) and G (3B).The 2D RNA structure is calculated by the KineFold program(http://kinefold.curie.fr/). Published editing sites are in blackcharacters (5) with the exception of site H which is out of thepresented 2D structure.

FIG. 4: Edited adenosines are conserved between Human and chimpanzee.Human intronic sequence depicted in FIG. 1 (base positions 5305 to 5736)was Blasted against chimpanzee build 2.1 genome database. Alignmentbetween the human sequence (upper raw, Query) and the Pan troglodytesreference sequence (ref|NW_001225252.1_Ptr15_WGA16816_2) (lower raw,Sbjct) is shown. Conserved adenosines are in red. Their names are inblack capital letters above the aligned sequences.

FIG. 5: Edited adenosines and intronic sequence are partially conservedbetween Human and Rhesus macaque. Human intronic sequence depicted inFIG. 1 (base positions 5305 to 5736) was Blasted against Rhesus monkeybuild 1.1 genome database, Alignment between the Human sequence (upperraw, Query) and the Macaca mulatta reference sequenceref|NW_001121189.1|Mmu7_WGA11353_1 (lower raw, Sbjct) is shown.Conserved adenosines are in red. Their names are in black capitalletters above the aligned sequences.

FIG. 6: In bold, underlined characters are presented the sequences ofthe Forward (FWD) and the Reverse (REV) unlabeled primers used for thefirst round of PCR (amplicon=495 bp). The two sequences corresponding tothe FAM-labeled FWD and VIC-labeled REV primers of the second nested PCR(amplicon=175 bp) are simply underlined. The sequence corresponding tothe RT primer is in italic, bold characters. The editing sites are shownwith their name above the sequence. New editing sites are in capitalletters. It must be noticed that with this set of primers, the H and Iediting sites can't be analyzed by CE-SSCP (see FIG. 12).

FIG. 7: Example of the limit of total RNA initial quantity necessary toobtain a constant evaluation of edited isoforms (Here the isoform B.Note that the editing profile presents an isoform B proportionindependent of the degree of dilution of the total RNA quantity used forthe initial RT until the smallest tested (62.5 ng).

FIGS. 8A-8C: Examples of editing profiles determined by CE from totalRNA extract of different Human tissues. On the left are presented thetypical analytical signals including FAM and VIC labeled strands (Seematerial and methods). The tables indicate the respective proportions ofeach isoform as % of the total of the expressed edited material.

FIGS. 9A-9B: Example of editing profile obtained from total RNA ofSH-SY5Y cells. In A the positive signals identify the mean CE signalobtained in control conditions (n=6 extracts). The negative signalscorrespond to Standard isoforms (See material and methods). In B thesignal has been amplified between the 600 and 700 points of the timebasis to give an example of the identification (here by the FAMfluorescence) of a peak which represented 1.54% of the total signal.

FIG. 10: Relationship between applied IFNα concentrations and the mean Aof variation of the B and non edited (NE) isoforms in the SH-SY5Y cells.Each point represents the mean ±SEM (n=8) of the individual valuesmeasured 48 hours after administration in the incubating medium of 0, 1,10, 100, 1000 and 10000 IU of IFNα.

FIG. 11: Correlation, in SH-SY5Y cells, between the relative quantities(RQ) of ADAR1a-150 induced by increased concentrations of IFNα and therelative increase in the proportions of isoform B in the editing profileof PDE8A RNA. The isoform B is defined as the isoform in which theedited site B is alone under the edited form.

FIG. 12: Schematic representation of the edited region of the PDE8Apre-mRNA. The edited sequence is located in intron 9 of the PDE8A gene.The sequences of the two labelled primers used for the nested PCR areshown and depicted as grey boxes in the schematic representation. Editedsites are indicated by vertical bars with their name above. Letters initalic (H and I) correspond to sites which cannot be analyzed with thisset of primers.

FIGS. 13A and 13B: Variations of the PDE8A pre-mRNA editing profile inthree cortical areas of controls subjects. A-Proportions of PDE8Apre-mRNA editing isoforms in Dorsoprefrontal Cortex (DPFCx), AnteriorCingulate Cortex (ACCx) and Entorhinal Cortex (ERCx) of controls (n=10for each area). Only editing isoforms with proportions higher than 3% intwo over the three brain areas are presented (B, ABC, Ne, BE, ABE, AB).Standard error of the mean (SEM) are shown (n=10). B-Ratios of editingisoform proportions between DPFCx, ACCx, and ERCx in controls.Individual values were log normal (LN) transformed to provide data witha normal distribution. For each considered isoform, all possibledifferences between individual controls of brain areas were calculated(n=100). The mean proportions ratios between brain areas were obtainedas exponential elevation of the above mentioned mean difference. For anabsence of variation the ratio=1. For significance, * stands for p≤0.05,** stands for p≤0.005, *** for p≤0.0005 and **** for p≤0.00005.

FIGS. 14A and 14B: The PDE8A pre-mRNA editing profile is significantlymodified in the ACCx of depressed suicides versus controls.A-Proportions of PDE8A pre-mRNA editing isoforms in Anterior CingulateCortex (ACCx) of controls and depressed suicides (n=10). Editingisoforms are those considered in FIG. 13 (B, ABC, Ne, BE, ABE, AB withproportions >3%). Standard error of the mean (SEM) are shown (n=10).B-Ratios of editing isoform proportions between depressed suicides andcontrols in ACCx. For each considered isoform, all possible differencesbetween LN (individual isoforms proportions) in controls of and insuicides compared to controls were calculated (n=100). The proportionsratios between suicides and controls were obtained by exponentialelevation of the above mentioned mean differences. For an absence ofvariation the ratio=1. For significance, * stands for p≤0.05, ** standsfor p≤0.005, *** for p≤0.0005 and **** for p≤0.00005.

FIG. 15: mRNA expressions of ADARs and PDE8A are altered in bloodsamples of suicide attempters. Suicide attempters were compared todepressed patients without any suicide attempt (n=25). The ADAR1a,ADAR1b, ADAR2 and PDE8A mRNA expressions were measured from total blood(PAXgene tubes) by the Q-PCR technique with the following specificprimers: HS01020780_m1; HS01017596_m1; HS00210762_m1 and HS00400174_m1respectively (Applied Biosystems References). Results were normalized bycomparison with respective values found in a reference pool of humanleucocytes total RNA. [reference genes:Glyceraldehyde-3Phospate-deshydrogenase (GAPDH) and β2 microglobuline(β2M)]. For significance, **** stands for p≤0.00005.

EXAMPLES Example 1: Material and Methods 1—Cell Culture, IFNα Treatment,Cells Lysis and RNA Extraction

The SH-SY5Y Human neuroblastoma cell line was purchased from ECACC (ref94030304, lot number 06H021). Cells were cultured in high glucose D-MEMmedium (Sigma, ref D6546) supplemented with 10% dialysed FCS (PAA, refA15-507, lot number A50708-0050), 2 mM Glutamine (Sigma, G7513) and a 1×mix of Antibiotic-Antimycotic Stabilized (Sigma, ref A5955) at 37° C.under a humidified atmosphere of 5% CO₂. The day preceeding hIFNαtreatment, SH-SY5Y cells were plated in 8 different 6-well plates at adensity of 7.10⁵ cells/well. On the next day, culture medium was removedand cells were incubated for 48 hours with hIFNα at the correctconcentration (PBL biomedical laboratories, ref 111001-1, lot number3734). On the day of the experiment, the hIFNα stock solution (10⁵I.U/ml in sterile 1× D-PBS stocked at −80° C.) was thawed on ice andthen extemporaneously diluted in D-MEM supplemented with FCS andAntibiotic-Antimycotic, at the final concentrations of 1, 10, 100, 1000and 1000 IU/ml. In each well, cells were treated with 2 ml of theworking solution. Aliquots of the stock-solution were used only once.For controls (vehicle), cells were treated with 2 ml of supplementedD-MEM. After 48 hours of incubation, medium was discarded and cellscorresponding to the 8 wells of an experimental condition were directlylysed in 600 μl of RET lysis buffer (Qiagen, RNeasy Plus mini Kit, ref74134) as described by furnisher.

RNA isolation and purification were carried out essentially as describedby manufacturer (Qiagen, RNeasy Plus Mini kit, ref 74134). Forhomogenization, cell lysates were first passed through QIAshredder spincolumns (Qiagen, ref 79656) placed in 2 ml collections tubes.Flow-throughs were then transfered to a gDNA Eliminator spin column ofthe RNeasy Plus Mini kit in order to eliminate remaining genomic DNA.RNAs were then purified on RNeasy spin columns and elated with 40 μl ofRNase-free water. Eluted RNAs were kept on ice for further experimentsor stocked at −30° C.

The quantity of total RNA obtained from each purification was measuredwith a Qubit Fluorometer (Invitrogen, ref Q32857) and the Quant-IT RNABR assay (Invitrogen, ref Q10211). The quality of RNAs was checked byloading 1 μg of the material on a native 1.5%agarose gel. The integrityof bands corresponding to 28S and 18S rRNA was verified for each sample.

2—Isolation of PBMCs from Human Total Blood and RNA Extraction

Blood samples (2×5 ml) were collected into heparinized tubes, pooled anddiluted with an equal volume of Ca²⁺ and Mg²⁺ free 1× PBS (PhosphateBuffer Saline) sterile solution. Two LeucoSep tubes (Greiner Bio-One,ref: 163 289 or 163 290) filled with 3 ml of pre-warmed separationmedium (Ficoll-Paque Plus, GE Healthcare Bio-Sciences AB, ref:17-1440-02) were centrifugated for 30 s at 1000g at room temperature.Half of the diluted blood volume was carefully poured into each of theseparation medium-containing LeucoSep tubes.

After 10 minutes of centrifugation at 1000g and room temperature, theenriched cell fractions were harvested and pooled(lymphocytes/PBMCs=white ring). The cells were then washed with 10 ml ofCa²⁺ and Mg²⁺ free 1× PBS sterile solution. After centrifugation 10minutes at 250 g the dry pellet was disrupted in 1 ml of TRIzol reagent(Invitrogen). The following phase separation and RNA precipitation stepswere performed according to manufacturer's instructions (TRizol Reagent,Invitrogen). The RNA pellet was washed twice with 1 ml of 75% ethanol,dried and resuspended in 50 μl of RNAse-free water. RNA concentrationswere determined with a Qubit Fluorometer (Invitrogen, Q32857) and theQuant-IT RNA BR assay (Invitrogen, ref Q10211).

3—Construction of Standard Editing Isoforms for CE-SSCP

Before cDNA synthesis 1 μg of total RNA (Human Blood PeripheralLeukocytes Total RNA, Clontech, ref 636580 [pool of 53 male/femaleCaucasians, ages: 20-50] and Human Brain Cerebral Cortex Total RNA,Clontech, ref 636561 [pool of 10 male/female Caucasians, ages: 20-68)was treated with 1 unit of RQ1 RNase-free, DNase (Promega, ref M610A)for 30 minutes at 37° C. The reaction was stopped by adding 1 μl of StopSolution (20 mM EGTA, Promega, ref part number M199A) and then heatedfor 10 minutes at 65° C. for both enzyme denaturation and RNAlinearization. RNA containing tubes were then immediately placed andkept on ice. DNase-treated RNAs were then reverse transcribed with theThermoscript RT-PCR system Plus Taq (Invitrogen, ref 11146-032) and thegene specific primer PDE8A-RT: 5′P-GTGGTAGGGAAAGCCAGGATG-3′OH (SEQ IDNO. 5) located in intron 9 of the Human PDE8A gene. The PCR reaction(final volume 50 μl) resulting in a 202 bp fragment, was carried out on2 μl of the reverse transcription products with 1 unit of Platinum PfxDNA polymerase (Invitrogen, ref 11708-013) and intron 9-specific primers(forward primer 5′P-CAACCCACTTATTTCTGCCTAG-3′OH (SEQ ID NO. 6) andreverse primer: 5′P-TTCTGAAAACAATGGGCACC-3′OH (SEQ ID NO. 7); finalconcentration 0.3 μM each). After a denaturing step at 95° C. for 5minutes, the PCR was brought to its final point after 35 cycles (30seconds at 95° C.; 30 seconds at 62° C. with a decreasing temperatureafter cycle 10 by 0.5° C. every 1 cycle, and 30 seconds at 68° C.), anda final elongation step of 2 minutes at 68° C. Aliquots (5 μl) of theamplification products were used to check the quantity and the qualityof amplicons on a 2% agarose analytic gel. The remaining 45 μl of eachPCR reaction were run on a preparative 2% agarose gel. Under longwave UVlight, agarose slices containing the PCR products were cut off and DNAwas then purified with the QIAquick gel extraction kit (Qiagen, ref28704). The purified PCR products were sent to GeneCust for cloning inthe pUC57 vector, and sequencing. One hundred and fifty clones comingfrom both tissue sources (cerebral cortex and leukocytes) weresequenced. Sequence analysis was performed at Biocortech and theoccurrence of each editing isoform quantified. Plasmids corresponding tothe different editing isoforms were then amplified and used as standardsin CE-SSCP experiments.

4—Reverse Transcription and Nested PCR

Human total blood RNA was extracted with the PAXgene Blood RNA kit 50(PreAnalytiX, ref 762174) according to manufacturer's instructions.Total RNA was treated with RQ1 RNase-free, DNase (Promega, ref M610A)for 30 minutes at 37° C. The reaction was stopped by adding 1 μl of StopSolution (20 mM EGTA, Promega, ref part number M199A) and then heatedfor 10 minutes at 65° C. for both enzyme denaturation and RNAlinearization. RNA containing tubes were then immediately placed andkept on ice. One microgram, 500 ng, 250 ng, 125 ng or 62.5 ng ofDNase-treated RNAs were reverse transcribed with the Thermoscript RT-PCRsystem Plus Taq (Invitrogen, ref 11146-032) and the gene specific primerPDE8A-RT: 5′P-GTGGTAGGGAAAGCCAGGATG-3′OH (SEQ ID NO. 5) located inintron 9 of the Human PDE8A gene. The first PCR reaction (final volume25 μl) resulting in a 495 bp fragment, was carried out on 1 μl of thereverse transcription products with 1 unit of Platinum Taq DNApolymerase (Invitrogen, ref 11146-032) and intron 9-specific unlabeledprimers (forward primer: 5P-GCTGAAGCCTTCCTTCTAAGG-3′OH (SEQ ID NO. 8)and reverse primer: 5P-CCTGGGTCAACTCTAGGTCC-3′OH (SEQ NO. 9); finalconcentration 0.3 μM each). After a denaturing step at 95° C. for 3minutes, the PCR was brought to its final point after 35 cycles (30seconds at 95° C., 30 seconds at 50° C., and 30 seconds at 72° C.), anda final elongation step of 2 minutes at 72° C. Products of this firstPCR were checked on a 2% agarose analytic gel and then diluted 1:100 forthe second round PCR. This second reaction (final volume 25 μl)resulting in a 175 bp fragment, was carried out on 1 μl of the 1:100dilutions with 1 unit of Platinum Pfx DNA polymerase (Invitrogen, ref11708-013) and intron 9-specific labeled primers (forward primer:FAM-5′P-CTAGGGAACCCTGTTTAGTCC-3′OH (SEQ ID NO. 10) and reverse primer:VIC-5′P-CAATGGGCACCAAAAAAGGG-3′OH (SEQ ID NO. 11); final concentration0.3 μM each). After a denaturing step at 94° C. for 4 minutes, the PCRwas brought to its final point after 35 cycles (30 seconds at 95° C. 30seconds at 50° C., and 30 seconds at 68° C.), and a final elongationstep of 2 minutes at 68° C. Aliquots (5 μl) of the amplificationproducts were used to check the quantity and the quality of amplicons ona 2% agarose analytic gel.

For other RNA sources (Total RNA from T-Helper/Inducer Lymphocytes(CD4-positive), Yorkshire Bioscience, ref N1121; Human Blood PeripheralLeukocytes Total RNA, Clontech, ref 636580; Human Brain Cerebral CortexTotal RNA, Clontech, ref 636561 and total RNA from PBMC or SH-SY5YNeuroblastoma cell line), 500 ng of total RNA were reverse transcribedand the resulting cDNAs amplified by nested-PCR as described above.

5—Quantification of ADAR1a-p150 mRNA Expression by Real-Time PCRAnalysis

In order to quantify levels of ADAR1a mRNA expression in SH-SY5Y cellsfirst-strand cDNA was synthesized by reverse transcription (as describedabove) and subjected to TaqMan quantitative Real-Time PCR analysis(Applied Biosystems). The probe and primers used for the quantitativePCRs were from Applied Biosystems (Gene Expression Assays,Assay-On-Demand):

-   ADAR1a: ref Hs 01020780_m1

Human GAPDH (product no. 4326317E; Applied Biosystems) was included ineach multiplex PCR as an internal control. Q-PCR and subsequent analysiswere performed with a 96-well block StepOnePlus real-time PCR system(Applied Biosystems). Quantitation of target gene expression in allsamples was normalized to GAPDH expression by the equationCt(target)−Ct(GAPDH)=ΔCt, where Ct is the threshold cycle number. Themean ΔCt value of samples from untreated cells was determined and usedas a reference point for the samples corresponding to IFNα treatedcells. Differences between untreated and treated cells, includingindividual variation were calculated by the equation ΔCt(individualtreated samples)−ΔCt(mean of untreated samples)=ΔΔCt. Changes in targetgene expression (n-fold) in each sample were calculated by 2^(−ΔΔCt),from which the means and standard deviations (SD) were derived.

6—Separation of Single-Strand cDNA Fragments by CapillaryElectrophoresis (CE-SSCP).

For the analysis of FAM- and VIC-labelled cDNA fragments by mean oftheir unique single-strand conformational polymorphism (SSCP), thefluorescent PCR products (1 μl of a 1:20 to 1:200 dilution) plusdeionized formamide (11 μl) were added to a mixture of migration andediting isoform standards (0.5 μl). The migration standards are PCRamplicons of different sizes labeled with the ROX fluorescent dye(Eurofins MWG operons). They are used for the calibration of theelectrophoresis migration in capillaries. Editing isoform standards(both FAM- and VIC-labelled)—whose construction has been describedabove—are used for unambiguous identification of the different editingisoforms present in the different samples. Before loading, the mixturesof samples and standards were denatured for 2 minutes at 90° C. and thenimmediately chilled on ice. Non-denaturing electrophoresis was carriedout in an ABI PRISM® 3100-Avant Genetic Analyzer (Applied Biosystems)through 80 cm-long capillaries filled with 7% “POP™ ConformationalAnalysis Polymer” (Applied Biosystems), 1× Tris-borate-EDTA and withoutglycerol. After a pre-run carried out at 15 kV for 3 min, samples wereinjected for 15 s at 2 kV, and electrophoresis was performed for 105 minat 15 kV, at a strictly controlled temperature of 24° C. In theseconditions, an individual retention time was obtained for each editingisoform. The procedures used for CE-SSCP analysis of RNA editing hasbeen extensively described in the article by Poyau and collaborators(16).

7—Identification and Relative Quantization of Each cDNA Form in aComplex Mixture.

Raw data obtained from the ABI PRISM® 3130x/t Genetic Analyzer wereextracted for signal processing by the PeakFit® (v4.11) software. Afterbase-line treatment and normalization of each electrophoresis profile(FAM- or VIC-labeled fragments) the relative abundance of the differentedited isoforms was quantified thanks to an in-house software allowingdeconvolution of the isoform and sample signals in an unique time basis.

8—Proteins Extractions and Western-Blot Analysis

After culture medium elimination, SH-SY5Y cells were washed two timeswith a phosphate buffer solution (PBS, Gibco Invitrogen Corporation),scraped and solubilized for 2 hr at 4° C. in solubilization buffercontaining 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% sodiumdeoxycholate, 10 mM Tris-HCl [pH 8.0] and supplemented with proteaseinhibitors (1 mM phenylmethylsulfonyl fluoride, and one tablet ofComplete™ mini protease inhibitors cocktail [Roche]). The lysate wasthen centrifugated for 10 min at 13,000×g at 4° C. to pellet celldebris. Proteins present in the supernatant (clear lysate) werequantified with a Qubit Fluorometer (Invitrogen, Q32857) and theQuant-IT Protein assay (Invitrogen, ref Q33211).

75 μg of the clear lysates corresponding to each experimental conditionwere resolved on 4-20% Tris-HCl gel (Bio-Rad, Criterion Precast Gels ref345-0033) at 100V for 3 hours.

Proteins (clear lysates from whole cell extracts) were then transferredonto nitrocellulose membrane (nitrocellulose transfer membrane ProtranBA 85, Schleicher and Schuell) using Towbin buffer (Towbin et al., 1979,PNAS, 76, 4350-4354) and a semi-dry electrotransfer device (Bio-Rad).After transfer, membranes were blocked, in 5% non-fat dried milk in TBST(10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.05% Tween 20) supplemented withsodium azide (0.1%) for 2 hr. The membrane was then incubated for 16 hrat room temperature with the primary antibody (Santa-Cruz, anti-PDE8A(C-15) ref se-17232) diluted 1:200 in the same buffer. After severalwashes with TBST, the blot was incubated with a Alexa Fluor 680anti-goat secondary antibody (Invitrogen, A21088) diluted 1:1000. Signalwas then read on an Odyssey machine (LiCor Biosciences).

9—CE-SSCP Method, Particularly for the Determination of the EditingProfile of the 5-HT2C Receptor

The method used for CE-SSCP determinations was already described forhuman samples (see patent PCT/EP 2008/057519 filed on Jun. 13, 2008 andpatent PCT/EP2009/067464 filed on Dec. 17, 2009).

Example II: The A to I Editing of PDE8A is Specific of More EvolvedPrimates Including Man

Interestingly, as shown in FIG. 4 all the adenosines described as editedin intron 9 of the PDE8A Human gene (A, B, C, D, E, F, and G) areconserved in the corresponding intronic sequence of chimpanzee. Theoverall identity is very high=428/432 (99%) and there is no gaps=0/432(0%) in the two sequences alignment, This very high similarity ofsequences, and potentially of secondary structures, between the twointrons strongly suggest that the adenosines residues could be edited inchimpanzee as they are in Human sequence.

In the Rhesus monkey sequence the adenosines are partially conserved,see FIG. 5. Actually, the A site of editing is not present in the Rhesussequence and the overall sequence homology is lower than the oneobserved with chimpanzee=399/433 (92%). Moreover 10 gaps/433 (2%) aredetected implying a lower conservation of the 2D RNA structure. SimilarBLAST analysis against mouse and rat genomes show that the sequence ofintron 9 of gene PDE8A is not conserved in these two other species.These results suggest that the potential editing of intronic sequence(intron 9) of gene PDE8A is limited to primates in mammals. Thefunctional reasons for this editing conservation are unknown but mayimply higher cerebral activities in primates and notably mooddisturbances. Finally the possible editing of sites in intron 9 of thePDE8A gene appears specific to some primate species with 8 alreadyidentified sites conserved in Human and Chimpanzee.

Example III: The Identification of the Editing Sites in this GenomicRegion has Been Completed by Cloning and Sequencing Technique Realizedon 150 Clones From Both Human Brain Cerebral Cortex and Leukocytes RNAExtracts (See Example 1: Materials and Methods)

These new edited sites are named I, J, K, L, M, N, and the correspondingexpressed edited isoforms are presented on the following table:

TABLE I New editing sites and corresponding edited isoforms in differenthuman tissues. Table I: New editing sites coordinates (underlined inbold) Editing sites Coordinates in intron 9 of PDE8A gene A 5505 B 5506C 5536 D 5538 E 5539 F 5548 G 5617 H 5468 I 5482 J 5500 K 5503 L 5544 M5572 N 5590

TABLE 2 Editing isoforms observed in Human leukocytes (150 clonessequenced). Editing isoforms >1% are in bold characters. Isoforms Numberof clones %/Total A 0 0.0 1 AB 2 1.3 1 ABDF 1 0.7 2 ABE 1 0.7 2 ABG 10.7 1 ABK 1 0.7 3 B 68 45.3 1 BC 10 6.7 1 BCD 2 1.3 2 BCE 2 1.3 2 BCF 21.3 2 BCG 1 0.7 2 BD 10 6.7 2 BDE 2 1.3 2 BDL 1 0.7 3 BE 4 2.7 2 BEF 21.3 2 BF 2 1.3 2 BFG 1 0.7 2 BG 3 2.0 2 BH 1 0.7 2 BK 1 0.7 3 D 1 0.7 2FGM 1 0.7 3 *ned 30 20.0 1 *ned: Non Edited isoform 1 Editing isoformsalready identified in PBMC 2 Editing isoforms identified in a pool ofleukocytes 3 Isoforms with new editing sites

TABLE 3 Editing Isoforms observed in Human brain cerebral cortex (150clones sequenced). Editing isoforms >1% are in bold characters. IsoformsNumber of clones %/Total A 0 0.0 1 AB 11 7.3 1 ABC 5 3.3 2 ABCDEFG 1 0.72 ABCEF 1 0.7 2 ABCG 1 0.7 2 ABDE 1 0.7 2 ABDEFG 1 0.7 2 ABDEG 1 0.7 2ABE 2 1.3 2 ABEF 2 1.3 2 ABEFG 3 2.0 2 ABEG 1 0.7 2 ABF 1 0.7 2 ABFG 10.7 2 ABFGI 1 0.7 3 ABG 2 1.3 2 ABN 1 0.7 3 B 64 42.7 1 BC 9 6.0 1BCDEFG 1 0.7 2 BCEG 1 0.7 2 BCFG 1 0.7 2 BCG 1 0.7 2 BD 3 2.0 2 BE 4 2.72 BEG 5 3.3 2 BF 3 2.0 2 BFG 2 1.3 2 BG 4 2.7 2 BH 1 0.7 2 BJ 1 0.7 3 M2 1.3 3 ned* 12 8.0 1 *ned: Non Edited isoform 1 Editing isoformsalready identified in PBMC 2 Editing isoforms identified in a pool ofcortex 3 Isoforms with new editing sites

An additional interest of this discovery was to allow the preparation ofstandards of each expressed edited isoforms in the brain and peripheralhuman tissues and in human 10 derived cell lines. It was realized bysubcloning RT-PCR products in the pUC57 vector as indicated in methodsection.

Example IV: The Conditions of the Precise Measurement of theDistribution of the Edited and Non Edited Isoforms of the PDE8A Pre-mRNAwere then Validated and Presented Here in Different Tissues or Cells asan Example

Thus, in Human tissues, the identification of these 14 editing sitescould conduct to a theoretical combination of 2¹⁴ pre-RNA isoforms andit was important to establish the degree of complexity of the editingprofile in different human tissues and human cell lines.

As typical examples the editing profile of PDE8A was identified and itsquantification validated in Human brain cerebral cortex RNA, in humanPeripheral Blood Mononuclear Cell (PBMC) total RNA, in Total blood RNAand in SH-SY5Y human cell line (Neuroblastoma derived) (see figures). Inthese cells the effect of interferon alpha, (a molecule known to inducesevere mood adverse effects in 20 to 50% treated patients (14,15) wasevaluated and allowed to demonstrate the interest of editing profilingto follow the alterations of the activity of editing enzymes. Startingfrom a total RNA extract the conditions of amplification of the genesequence including the 7 edited sites were tested to allows a limit ofinitial total RNA quantity below 70 ng, and to obtained the bestcombination of forward and reverse specifically labeled single strandsallowing specific separation of expressed isoforms by capillaryelectrophoresis. Finally, to obtain the best sensitivity (adequateresults for 62 ng of starting RNA material for RT) and adequateidentification of expressed isoforms the following two steps nested. PCRwas validated using the following primers:

Thus, the defined primers are:

-   1st PCR/unlabeled primers:

PDE8A-1REV (SEQ ID NO. 12) GGACCTAGAGTTGACCCAGG PDE8A-1FWD (SEQ ID NO.13) GCTGAAGCCTTCCTTCTAAGG

-   2d PCR/FAM FWD labeled and VIC REV labeled primers:

PDE8A-2Rev VIC (SEQ ID NO. 11) CAATGGGCACCAAAAAAGGG PDE8A-2FowFAM (SEQID NO. 10) CTAGGGAACCCTGTTTAGTCC

This choice was the result from a specific screening in order todetermine the best sensitivity (limit of use of initial concentrationsof total RNA in a given sample) and the best reliability of the PCRsproducts and the best length of the single strand to allows a goodseparation of the majority of the expressed isoforms in a given tissue(see FIGS. 7, 8A-8C and 9A-9B).

TABLE 4 SH-SY5Y Foetal Veal Serum Isoforms Mean % SEM A 0.00 AB 0.00 ABG1.54 0.21 B 12.24 0.42 BC 0.00 ned 86.22 0.31 SUM 100.00 ned: Non Editedisoform

Example V: Identification of the Editing Isoforms Profile as a ReliableIndex of the Alterations of the Activities of the Editing Enzymes

As an example the alteration produced by the pharmacological modulationof the expression of the ADAR1a-150 isoenzyme was tested on SH-SY5Y cellline. The results are summarized in FIG. 10.

Thus is demonstrated that, in this cell line, the positive variation ofthe Isoform B expression is clearly concentration dependable. The nedisoform symmetrically negatively decrease in proportion indicating thatthe isoform B is mainly if not exclusively produced by the ADAR1a-p150since its variation closely correlates with the variation of expressionof this editing enzyme (See FIG. 11). The EC 50% which was calculatedfrom this study was in the same order of value than the EC50% alreadydemonstrated for the editing of the 5-HT2cR in the same conditions inthe same cell line.

Example VI: PDE8A is Expressed in the Brain and an Editing Profile canbe Observed

The PDE8A RNA edited isoforms characterized by their edited sites canthus be identified from total brain RNA following a process which can besummarized as follows:

Material and Methods

Brains were collected at autopsy, sectioned coronally, flash-frozen andstored at −80° C. until dissection. Cerebral trauma, central nervoussystem pathology, alcoholism or drug use disorder were exclusioncriteria. Brain samples were assayed and analyzed by personal blinded tothe cause of death. pH was measured in the cerebellum. A psychologicalautopsy was used to obtain DSM-IV Axis I and II diagnoses.

Control subjects (n=10) died from causes other than suicide and did notmeet criteria for any Axis I during their lifetime. Suicides (n=10) metcriteria of the Columbia classification of suicidal behavior.

Brains Regions and RNA Extraction

The dorsolateral prefrontal (Brodmann area 9, DPFCx), anterior cingulate(Brodmann area 24, ACCx) and entorhinal (Brodmann area 28/34, ERCx)cortex were selected as regions of interest because they have beenconsistently implicated as being altered in depression and/or suicide.Wet weight of tissue (mean±SEM) was 80±4 mg in DPFCx, 84±4 mg in ACCxand 80±5 mg in ERCx.

Total RNA was isolated from affinity columns using RNeasy lipid. TissueMini Kit (Qiagen). Genomic DNA contamination was removed by on-columnDNase digestion. The yield of total RNA (absorbance at 260 nm) rangedfrom 12 to 57 μg. The total amount of RNA used for the reversetranscription of each sample was uniformly 1 μg.

Reverse Transcription, Nested-PCR and Identification and RelativeQuantification of Each Brain Sample

As described in previous sections of material and methods.

Statistical Analysis

Individual values were log normal (LN) transformed to provide data witha normal distribution. Each expressed isoform, each corticalinvestigated brain region, each group of subjects was then preciselyindexed and were used as independent variables. The all possibledifferences between individual LN transformed proportions of theisoforms measured in controls individuals in the 3 regions (DPFCx, ACCxand ERCx) or in suicides and controls individuals in one particularregion (the ACCx in this example) were analysed by discriminant ANOVAand adequate post hoc analysis (Scheffé test). The p values are givenfor no differences between regions or subjects groups and thedifferences are considered as significant for p≤0.05.

Results

The editing profile of the PDE8A pre-mRNA is significantly different inthe three cortical regions (FIGS. 13A and B) and altered in depressedsuicides versus controls (FIG. 14 as an example)

First, we have established the editing profile of the PDE8A pre-mRNA inthree brain areas of the controls subjects [Dorsolateral PrefrontalCortex (DPFCx), Anterior Cingulate Cortex (ACCx) and Entorhinal Cortex(ERCx)]. Six editing isoforms with proportions higher than 3% in atleast one of the three brain areas (B, ABC, Ne, BE, ABE, AB) wereanalyzed (see FIG. 13A). For each of these isoforms, the ratios of theirproportions in the DPFCx, the ACCx and the ERCx are presented in FIG.13B. Significant variations were observed between the different areasand notably in the case of the AB iso form which is differentiallyexpressed in the three cortical areas (compare DPFCx vs ERCx, and ACCxvs ERCx). These results evidenced the fine regulation of the PDE8Apre-mRNA editing in the brain of controls subjects depending on theidentity and functional steady state of the cell networks which areusing PDE8A as a metabotropic regulator.

In a second step the proportions of the same isoforms were comparedbetween controls subjects and depressed suicides in the three corticalareas. As an example the proportions of the six isoforms in the ACCx ofdepressed suicides versus controls is presented in FIG. 14A. For each ofthe six isoforms, the ratios of their proportions in depressed suicidesversus controls is shown in FIG. 14B. The editing profile of the PDE8Apre-mRNA is significantly modified in the ACCx of depressed suicideswith editing isoforms up and down regulated likely in different cellcompartments.

Example VI: mRNA Expressions of ADARs and PDE8A are Altered in BloodSamples of Suicide Attempters

PDE8A is present in total blood like the editing enzymes:

It was thus interesting to approach the possible alteration of theexpression of editing enzymes and of the PDE8A mRNA to see if thistarget of the RNA editing was altered as a result of a particularsuicide risk. An example of the alteration of both editing enzymesADAR1a, ADAR1b and ADAR2 and of one of their target PDE8A expression hasbeen demonstrated by measuring these markers in two populations ofpatients. The first one was depressed subjects tested just after asuicide attempt (considered as a particular suicide risk population(SuicideAtt), the second one used as control was included as depressed(MD) without any suicide attempt. The initial result is summarized onFIG. 15, obtained after determination of the steady state of thesebiomarkers RNA concentrations in total blood.

Example of Alteration of PDE8A mRNA Expression Associated withSuicidality in Blood of Suicide Attempters

The alteration of levels of expression of editing enzymes and PDE8A wasobserved in suicide attempters compared to depressed patients withoutany suicide attempt (n=25). ADAR1a, ADAR1b ADAR2 and PDE8A mRNA weremeasured from total blood (sampled in PAXgene RNA tubes). ADAR1a,ADAR1b, ADAR2 and PDE8A mRNA steady state concentrations were measuredby QPCR from 500 ng of total RNA samples by using specific printers(HS01020780_m1; HS01017596_m1; HS00210762_m1 and HS00400174_m1respectively, Applied Biosystems references) and normalized bycomparison with respective values found in a reference pool of humanleucocytes total RNA. [reference genes:Glyceraldehyde-3Phospate-deshydrogenase (GAPDH) and β2 microglobuline(β2M)]. We note the highly significant alteration of editing occuring insuicide attempters.

Finally Brain PDE8A pre-mRNA editing profile is modified in suicidesvictims. On the other hand, editing enzymes and PDE8A expressions arealtered in the blood of suicide attempters. This particular regulatorycapacity which is unique in Man and pre-Human primates represents aparticularly interesting way to evaluate by blood testing suicide risk.

This example illustrates the strong alteration of the editing process inrelation with the suicide risk. The PDE8A is concerned as well as theediting enzymes and it can be suggested that the editing of a non codingsequence of this target could be directly or indirectly involved in theregulation of the expression of this target.

REFERENCES

-   1—Jin Billy Li, Erez Y, Levanon, Jung-Ki Yoon, John Auch, Bin Xie,    Emilie LeProust, Kun Zhang, Yuan Gao, George M. Church: Genome-Wide    Identification of Human RNA Editing Sites by DNA Parallel DNA    capturing and Sequencing. Science 2009, 324: 1210-1213.-   2—Tim D. Werry, Richard Loiacono, Patrick M. Sexton, Arthur    Christopoulos: RNA editing of the serotonin 5-HT2c receptor and its    effects on cell signaling, pharmacology and brain function.    Pharmacol. Ther. 2008, 119: 7-23.-   3—Peter H. Seeburg, Miyoko Higuchi, Rolf Sprengel: RNA editing of    brain glutamate receptor channels: mechanism and physiology. Brain    Res. Reviews 1998, 26: 217-229.-   4—Daniel P. Morse, P. Joseph Aruscavage, and Brenda L. Bass: RNA    hairpins in noncoding regions of human brain and Caenorhabditis    elegans-mRNA are edited by adenosine deaminases that act on RNAs.    Proc. Natl. Acad. Sci. USA 2002, 99: 7906-7911.-   5—Robert J. Orlowski, Kenneth S. O'Rourke, Irene Olorenshaw,    Gregory A. Hawkins, Stefan Maas and Dama Laxminarayana: Altered    editing in cyclic nucleotide phosphodiesterase 8A1 gene transcripts    of systemic lupus erythematosus T lymphocytes. Immunology: 2008,    125: 408-419.-   6—M. Ohman: A-to-I editing challenger or ally to the microRNA    process. Biochimie 2007, 89: 1171-1176.-   7—S Dracheva, N Patel, D A Woo, S M Marcus, L J Siever, and V    Haroutunian: Increased serotonin 2C receptor mRNA editing: a    possible risk factor for suicide. Mol. Psychiatry 2008, 13:    1001-1010.-   8—Peter Holmans, George S. Zubenko, Raymond R. Crowe, J. Raymond    DePaulo Jr., William A. Scheftner, Myrna M. Weissman, Wendy N.    Zubenko, Sandra Boutelle, Kathleen Murphy-Eberenz, Dean McKinnon,    Melvin G. McInnis, Diana H. Marta, Philip Adams, James A. Knowles,    Madeleine Gladis, Jo Thomas, Jenifer Chellis, Erin B. Miller, and    Douglas F. Livinson: Genomewide Significant Linkage to Recurrent,    Early-Onset major Depressive Disorder on Chromosome 15q. Am. J. Hum.    Genet. 2004, 74: 1154-1167.-   9—Peter Holmans, Myrna M. Weissman, George S. Zubenko, William A.    Scheftner, Raymond R. Crowe, J. Raymond DePaulo Jr., James A.    Knowles, Wendy N. Zubenko, Kathleen Murphy-Eberenz, Diana H. Marta,    Sandra Boutelle, Melvin G. McInnis, Philip Adams, Madeleine Gladis,    Jo Steele, Erin B. Miller, James B. Potash, Dean F. McKinnon, and    Douglas F. Livinson: Genetic of Recurrent Early-Onset Major    Depression (GenRED): Final Scan Report. Am. J. Psychiatry. 2007,    164: 248-258.-   10—Peter McGuffin, Jo Knight, Gerome Breen, Shyama Brewster,    Peter R. Boyd, Nick Craddock, Mike Gill, Ania Korszun, Wolfgang    Maier, Lefkos Middleton, Ole Mors, Michael J. Owen, Julia Perry,    Martin Preisig, Theodore Reich, John Rice, Marcella Rietschel, Lisa    Jones, Pak Sham, and Anne E. Farmer: Whole genome linkage scan of    recurrent depressive disorder from the depression network study.    Hum. Mol. Genet. 2005, 14: 3337-3345.-   11—Yu Feng, Agnes Vetro, Enito Kiss, Krisztina Kapornai, Gabriella    Daroczi, Laszlo Mayer, Zsuzsana Tamas, Ildiko Baji, Julia Gadoros,    Nicole King, James L. Kennedy, Karen Wigg, Maria Kovacs, Cathy L.    Barr: Association of the Neurotrophic Tyrosine Kinase Receptor 3    (NTRK3) Gene and Childhood-Onset Mood Disorders. Am. J. Psychiatry    2008, 154: 610-616.-   12—Ranjana Verna, Peter Holmans, James A. Knowles, Deepak Grover,    Oleg V. Evgrafov, Raymond R. Crowe, William A. Scheftner, Myrna M.    Weissman, J. Raymond DePaulo Jr., James B. Potash, and Douglas F.    Levinson: Linkage Desiquilibrium Mapping of a Chromosome 15q25-26    Major Depression Linkage Region and Sequencing of NTRK3. Biol.    Psychiatry 2008, 63: 1185-1189.-   13—Peng Wang, Ping Wu, Robert W. Egan, M. Motasim Billah: Human    phosphodiesterase 8A splice variants: cloning, gene organization,    and tissue distribution. Gene 2001, 280: 183-194.-   14—Charles L. Raison, Andrey S. Borisova, Matthias Majer, Daniel F.    Drake, Giuseppe Pagnoni, Bobbi J. Woolwine, Gerald J. Vogt, Breanne    Massung, and Andrew H. Miller: Activation of Inflamatory Pathways by    Interferon-alpha: Relationship to Monoamines and Depression. Biol.    Psychiatry 2009, 65: 296-303.-   15—Weidong Yang, Qindge Wang, Stephen J. Kanes, John M. Murray,    Kazuko Nishikura: Altered RNA editing of serotonin 5-HT2c receptor    induced by interferon: implications for depression associated with    cytokine therapy. Mol. Brain Res. 2004, 124: 70-78.-   16—Alain Poyau, Laurent Vincent, Hervé Berthommé, Catherine Paul,    Brigitte Nicolas, Jean-François Pujol, Jean-Jacques Madjar:    Identification and relative quantification of adenosine to inosine    editing in serotonin 2c receptor mRNA by CE. Electrophoresis 2007,    28: 2843-2852.

1-34. (canceled)
 35. An in vitro method, comprising: a) providing abiological subject sample comprising Peripheral Blood Mononuclear Cells(PBMC), said PBMC expressing the editing enzymes ADAR1a, ADAR1b andADAR2, and the phosphodiesterase subtype 8A (PDE8A); b) preparing acellular RNA extract from the subject sample; and c) determining theediting profile of the PDE8A pre-mRNA in said cellular RNA extract,wherein determining the editing profile of the PDE8A pre-mRNA comprisesdetecting the ned (non edited isoform) and B isoforms.
 36. The methodaccording to claim 35, further comprising measuring the expression ofsaid editing enzymes ADAR1a, ADAR1b and ADAR2 in the subject sample. 37.The method according to claim 35, further comprising measuring theexpression of mRNAs encoding said editing enzymes ADAR1a, ADAR1b andADAR2 in the cellular RNA extract.
 38. The method according to claim 36,wherein the expression of said editing enzymes ADAR1a, ADAR1b and ADAR2is measured by measuring expression of mRNAs encoding said editingenzymes or by measuring expression of said editing enzyme proteins. 39.The method according to claim 36, wherein expression of said editingenzymes ADAR1a, ADAR1b and ADAR2 in the subject sample is measuredquantitatively.
 40. The method according to claim 35, wherein thebiological sample is a blood sample comprising white cells.
 41. Themethod according to claim 35, wherein the step c) of determining theediting profile of the PDE8A pre-mRNA further comprises detecting anisoform selected from I, J, K, L, M and N.
 42. The method according toclaim 35, wherein the step c) of determining the editing profile of thePDE8A pre-mRNA further comprises detecting an isoform selected from AB,ABC, ABE, ABEF, ABEFG, ABG, BC, BD, BE, BEG, BF, BFG, BG, and M.
 43. Themethod according to claim 35, wherein the step c) of determining theediting profile of the PDE8A pre-mRNA comprises detecting the ned, B andAB isoforms.
 44. The method according to claim 43, wherein the step c)of determining the editing profile of the PDE8A pre-mRNA comprisesdetecting the ned, B, AB and BC isoforms.
 45. The method according toclaim 35, wherein the editing profile of the PDE8A pre-mRNA isdetermined by a process comprising performing a reverse transcriptionreaction on the cellular RNA extract and performing a nested type PCRcomprising two rounds of PCR on the product of the reversetranscription, and wherein: a) the first round of PCR is carried out bythe following sets of primers: Forward: PDE8A-1FWD (SEQ ID NO. 13)GCTGAAGCCTTCCTTCTAAGG, Reverse: PDE8A-1REV (SEQ ID NO. 12)GGACCTAGAGTTGACCCAGG,

and and wherein b) the second round of PCR is carried out by thefollowing set of primers: Forward: PDE 8 A-2Fwd FAM (SEQ ID NO. 10)CTAGGGAACCCTGTTTAGTCC, Reverse: PDE8A-2Rev VIC (SEQ ID NO. 11)CAATGGGCACCAAAAAAGGG.


46. The method according to claims 35, wherein ADARs specific isoformsare determined in the method, and wherein the pair of primers specificfor the human ADAR mRNA PCR amplification are selected from the groupconsisting of: for ADAR1-150 isoform mRNA amplification: Forward: (SEQID NO. 14) 5′-GCCTCGCGGGCGCAATGAATCC-3′, Reverse: (SEQ ID NO. 15)5′-CTTGCCCTTCTTTGCCAGGGAG-3′,

and for ADAR1-110 isoform mRNA amplification: Forward: (SEQ ID NO. 16)5′-CGAGCCATCATGGAGATGCCCTCC-3′, Reverse: (SEQ ID NO. 17)5′-CATAGCTGCATCCTGCTTGGCCAC-3′,

and for ADAR2 mRNA amplification: Forward: (SEQ ID NO. 18)5′-GCTGCGCAGTCTGCCCTGGCCGC-3′, Reverse: (SEQ ID NO. 19)5′-GTCATGACGACTCCAGCCAGCAC-3′.

-3′ (SEQ ID NO. 19).
 47. The method of claim 35 wherein the subject is adepressed patient or a suicide attempter.
 48. The method of claim 35wherein the editing profile of the PDE8A pre-mRNA in said cellular RNAextract matches the editing profile of the PDE8A pre-mRNA for normalpatients
 49. The method of claim 35 wherein the editing profile of thePDE8A pre-mRNA in said cellular RNA extract matches the editing profileof the PDE8A pre-mRNA for a patient diagnosed with a pathology selectedfrom the group consisting of psychiatric disorders, mental disorders,schizophrenia, depression, Bipolar disease, suicide or abnormal feedingbehaviour, Mild Cognitive Impairement (MCI), Epilepsia, Alzheimer andChronical pain syndromes
 50. The method of claim 35 wherein the editingprofile of the PDE8A pre-mRNA in said cellular RNA extract matches theediting profile of the PDE8A pre-mRNA for a patient undergoing treatmentfor a pathology selected from the group consisting of psychiatricdisorders, mental disorders, schizophrenia, depression, Bipolar disease,suicide or abnormal feeding behaviour, Mild Cognitive Impairement (MCI),Epilepsia, Alzheimer and Chronical pain syndromes.